Bifunctional detection agents having a polymer covalently linked to an MRI agent and an optical dye

ABSTRACT

The invention provides bifunctional detection agents comprising optical dyes covalently linked to at least one magnetic resonance image (MRI) contrast agent. These agents may include a linker, which may be either a coupling moiety or a polymer.

The U.S. Government has certain rights in this invention pursuant togrant number DA 08944 from National Institutes of Health.

FIELD OF THE INVENTION

The invention relates to optically active magnetic resonance imagingagents.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) is a clinical diagnostic and researchprocedure that uses a high-strength magnet and radio-frequency signalsto produce images. The most abundant molecular species in biologicaltissues is water. It is the quantum mechanical "spin" of the waterproton nuclei that ultimately gives rise to the signal in imagingexperiments. In MRI the sample to be imaged is placed in a strong staticmagnetic field (1-12 Tesla) and the spins are excited with a pulse ofradio frequency (RF) radiation to produce a net magnetization in thesample. Various magnetic field gradients and other RF pulses then act onthe spins to code spatial information into the recorded signals. MRI isable to generate structural information in three dimensions inrelatively short time spans.

There is rapidly growing body of literature demonstrating the clinicaleffectiveness of paramagnetic contrast agents; currently there are atleast eight different contrast agents in clinical trials or in use.These agents provide further contrast, and thus enhanced images,wherever the contrast agent is found. For example, the approved contrastagents outlined below may be injected into the circulatory system andused to visualize vascular structures and abnormalities, amongst otherthings. The capacity to differentiate regions or tissues that may bemagnetically similar but histologically different is a major impetus forthe preparation of these agents.

The lanthanide atom Gd(III), has generally been chosen as the metal atomfor contrast agents because it has a high magnetic moment (μ² =63BM²), asymmetric electronic ground state, (S⁸), the largest paramagnetic dipoleand the greatest paramagnetic relaxivity of any element. Gd(III) isrendered nontoxic by chelation. To date, a number of chelators have beenused, including diethylenetriaminepentaacetic (DTPA), 1,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid (DOTA), andderivatives thereof. See U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553,5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al., Invest.Radiol. 25:S53 (1990).

The stability constant (K) for GD(DTPA) is very high (logK=22.4) and ismore commonly known as the formation constant (the higher the logK, themore stable the complex). This thermodynamic parameter indicates thefraction of Gd(III) ions that are in the unbound state will be quitesmall and should not be confused with the rate (kinetic stability) atwhich the loss of metal occurs. The water soluble Gd(DTPA)-chelate isstable, nontoxic, and one of the most widely used contrast enhancementagents in experimental and clinical imaging research. It was approvedfor clinical use in adult patients in June of 1988. It is anextracellular agent that accumulates in tissue by perfusion dominatedprocesses. Image enhancement improvements using Gd(DTPA) are welldocumented in a number of applications (Runge et al., Magn, Reson. Imag.3:85 (1991); Russell et al., AJR 152:813 (1989); Meyer et al., Invest.Radiol. 25:S53 (1990)) including visualizing blood-brain barrierdisruptions caused by space occupying lesions and detection of abnormalvascularity. It has recently been applied to the functional mapping ofthe human visual cortex by defining regional cerebral hemodynamics(Belliveau et al., (1991) 254:719).

Another chelator used in Gd contrast agents is the macrocyclic ligand1,4,7, 10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA). TheGd-DOTA complex has been thoroughly studied in laboratory testsinvolving animals and humans. The complex is conformationally rigid, hasan extremely high formation constant (logK=28.5), and at physiologicalpH possess very slow dissociation kinetics. Recently, the GdDOTA complexwas approved as an MRI contrast agent for use in adults and infants inFrance and has been administered to over 4500 patients.

Another technique for imaging cells, frequently used in developmentalbiology, uses optical dyes, i.e. photoluminescent compounds, tovisualize both subcellular and extracellular structures, as well asdevelopmental cell lineages.

It is an object of the invention to provide bifunctional detectionagents that can simultaneously behave as both an optical dye as well asan MRI contrast agent. Such agents can be visualized using either MRIand common optical (photoluminescent) techniques.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides bifunctional detection agents comprising optical dyescovalently linked to at least one magnetic resonance image (MRI)contrast agent. These agents may include a linker, which may be either acoupling moiety or a polymer. These bifunctional detection agentsinclude agents having the structure depicted below: ##STR1## wherein Xis a coupling moiety and R is a substitution moiety.

The invention also provides bifunctional detection agents having thestructure: ##STR2## wherein X is a coupling moiety and R is asubstitution moiety.

The invention additionally provides bifunctional detection agents havingthe structure: ##STR3##

The invention further provides bifunctional detection agent comprising apolymer covalently linked to at least one optical dye and at least oneMRI contrast agent. The polymer may comprise a polyamino acid. Thepolymer may have a molecular weight of less than 40 kD, 25 kD, 15 kD, or10 kD.

The invention further provides methods of visualizing cells and tissuescomprising the administration of the bifunctional detection agents withfluorescence and MRI detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of GABAR (Gadolinium amino benzyl rhodamine)as described in the examples.

FIG. 2 shows the synthesis of GRP (Gadolinium rhodamine polylysine) asdescribed in the examples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides bifunctional detection agents thatfunction as both magnetic resonance imaging (MRI) contrast agents aswell as optically detectable agents or dyes.

In a preferred embodiment, the bifunctional detection agents comprise anMRI contrast agent covalently linked to a optical dye.

By "MRI contrast agent " herein is meant a molecule that can be used toenhance the MRI image. As is known in the art, MRI contrast agentsgenerally comprise a paramagnetic metal ion bound to a chelator. By"paramagnetic metal ion", "paramagnetic ion" or "metal ion" herein ismeant a metal ion which is magnetized parallel or antiparallel to amagnetic field to an extent proportional to the field. Generally, theseare metal ions which have unpaired electrons; this is a term understoodin the art. Examples of suitable paramagnetic metal ions, include, butare not limited to, gadolinium III (Gd+3 or Gd(III)), iron III (Fe+3 orFe(III)), manganese II (Mn+2 or Mn(II), yttrium III (Yt+3 or Yt(III)),dysprosium (Dy+3 or Dy(III)), and chromium (Cr(III) or Cr+3). In apreferred embodiment the paramagnetic ion is the lanthanide atomGd(III), due to its high magnetic moment (U² =63BM2), a symmetricelectronic ground state (S8), and its current approval for diagnosticuse in humans.

In addition to the metal ion, the MI contrast agent usually comprise achelator. Due to the relatively high toxicity of many of theparamagnetic ions, the ions are rendered nontoxic in physiologicalsystems by binding to a suitable chelator. The chelator utilizes anumber of coordination atoms at coordination sites to bind the metalion. As is more fully described below, the replacement of a coordinationatom with a functional moiety to allow covalent attachment of the MRIcontrast agent to a linker or optical dye may render the metal ioncomplex more toxic by decreasing the half-life of dissociation for themetal ion complex. Thus, in a preferred embodiment, a site other than acoordination site is preferably used for covalent attachment. However,for some applications, e.g. analysis of tissue and the like, thetoxicity of the metal ion complexes may not be of paramount importanceand thus covalent attachment via a coordination site is appropriate.Similarly, some metal ion complexes are so stable that even thereplacement of one or more additional coordination atoms with a blockingmoiety does not significantly effect the half-life of dissociation. Forexample, both DTPA and DOTA, described below, are extremely stable whencomplexed with Gd(III). Accordingly, one or several of the coordinationatoms of the chelator may be replaced with one or more functionalmoieties for covalent attachment without a significant increase intoxicity.

There are a large number of known macrocyclic chelators or ligands whichare used to chelate lanthanide and paramagnetic ions. See for example,Alexander, Chem. Rev. 95:273-342 (1995) and Jackels, Pharm. Med. Imag,Section III, Chap. 20, p645 (1990), expressly incorporated herein byreference, which describes a large number of macrocyclic chelators andtheir synthesis. Similarly, there are a number of patents which describesuitable chelators for use in the invention, including U.S. Pat. Nos.5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704,5,262,532, and Meyer et al., Invest. Radiol. 25:S53 (1990), all of whichare also expressly incorportated by reference. There are a variety offactors which influence the choice and stability of the chelate metalion complex, including enthalpy and entropy effects (e.g. number, chargeand basicity of coordinating groups, ligand field and conformationaleffects, etc.). In general, the chelator has a number of coordinationatoms which are capable of binding the metal ion. The number ofcoordination atoms, and thus the structure of the chelator, depends onthe metal ion. Thus, as will be understood by those in the art, any ofthe known paramagnetic metal ion chelators or lanthanide chelators canbe easily modified using the teachings herein to add a functional moietyfor covalent attachment to an optical dye or linker.

Preferred MRI contrast agents include, but are not limited to,1,4,7,10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA),diethylenetriaminepentaacetic (DTPA),1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraethylphosphorus (DOTEP),1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (Do3A) andderivatives thereof (see U.S. Pat. No. 5,188,816, 5,358,704, 4,885,363,and 5,219,553, hereby expressly incorporated by reference).

As is described herein, in a preferred embodiment the MRI agent issubstituted at any number of possible positions with a functional groupto facilitate the covalent attachment of the optical dye or linker suchas a coupling moiety or polymer. For example, when the contrast agent isDOTA, a preferred embodiment utilizes any one of the R sites ofstructure 1 as the site of covalent attachment. ##STR4##

In an additional embodiment, one of the carboxylic acid chelating "arms"(i.e. a coordination atom) of DOTA may also be used as the site ofcovalent attachment, as depicted in Structure 2 (unsubstituted DOTA,although substituted compounds as also included: ##STR5##

In this embodiment, either a carbonyl (i.e. as depicted with R inStructure 2), an ester linkage (i.e. as depicted with R₁), or directlinkage to the nitrogen atom (as depicted in R₂ and R₃) may be formed,depending on the type of coupling chemistry used, as is more generallyoutlined below. As will be appreciated by those in the art, similarsubstitutions may be done with DTPA and DOTEP (the unsubstituted formsof which are depicted in Structures 3 and 4, respectively, although asoutlined above in Structure 1, the carbon atoms of DTPA and DOTEP or anyother MRI agent of use herein may be substituted with R groups):##STR6##

Other suitable Gd(III) chelators are described in Alexander, supra,Jackels, supra, Lauffer et al., supra, U.S. Pat. Nos. 5,155,215,5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532, andMeyer et al., Invest. Radiol. 25:S53 (1990), among others.

As will be appreciated by those in the art, the R sites outlined hereinmay also comprise additional substitution groups, in addition to the Rsite that is used for covalent attachment. Suitable substitution groupsinclude a wide variety of groups, as will be understood by those in theart. For example, suitable substitution groups include substitutiongroups disclosed for DOTA and DOTA-type compounds in U.S. Pat. Nos.5,262,532, 4,885,363, amd 5,358,704. These groups include hydrogen,alkyl and aryl groups, substituted alkyl and aryl groups, amino groups,hydroxy groups, thiol groups, phosphorus moieties, etc. Preferredsubstitution groups include hydrogen. As will be appreciated by thoseskilled in the art, each position outlined herein may have two R groupsattached (R' and R"), although in a preferred embodiment only a single Rgroup is attached at any particular position. Thus, for example, the MRIcontrast agents utilized in the invention may be substituted at any oneof the R positions with moieties to confer or neutralize charge, alterthe hydrophobicity or hydrophilicity, or alter the molecular weight. Thelarger the molecule, the slower it rotates in solution and therelaxivity increases.

Chelators for use with other metals are known. For example, suitablechelators for Fe(III) ions are well known in the art. See Lauffer etal., J. Am. Chem. Soc. 109:1622 (1987); Lauffer, Chem. Rev. 87:901-927(1987); and U.S. Pat. Nos. 4,885,363, 5,358,704, and 5,262,532, allwhich describe chelators suitable for Fe(III). Suitable chelators forMn(II) ions are also well known in the art; see for example Lauffer,supra, and U.S. Pat. Nos. 4,885,363, 5,358,704, and 5,262,532. Suitablechelators for Yt(III) ions include, but are not limited to, DOTA andDPTA and derivatives thereof (see Moi et al., J. Am. Chem. Soc.110:6266-6267 (1988)) and those chelators described in U.S. Pat. No.4,885,363 and others, as outlined above. Chelators for Dy+3 (Dy(III))are also known in the art and described in the references cited herein.

The MRI contrast agents are covalently attached to an optical dye toform the bifunctional detection agents of the invention. By "opticaldye" herein is meant a photoluminescent compound. That is, a compoundthat will emit detectable energy after excitation with light. In apreferred embodiment, the optical dye is fluorescent; that is, uponexcitation with a particular wavelength, the optical dye with emit lightof a different wavelength; such light is typically unpolarized. In analternative embodiment, the optical dye is phosphorescent.

Preferred optical dyes include, but are not limited to, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, and Texas Red.

Suitable optical dyes are described in the 1989-1991 Molecular ProbesHandbook by Richard P. Haugland, hereby expressly incorporated byreference.

In a preferred embodiment, the optical dye is functionalized tofacilitate covalent attachment. Thus, a wide variety of optical dyes arecommercially available which contain functional groups, including, butnot limited to, isothiocyanate groups, amino groups, haloacetyl groups,maleimides, succinimidyl esters, and sulfonyl halides, all of which maybe used to covalently attach the optical dye to a second molecule, suchas the MRI contrast agents or linkers used in the present invention.

The choice of the functional group of the optical dye will depend on thesite of attachment to the MRI contrast agent or linker, as outlinedbelow.

The covalent attachment may be either direct or via a linker. In oneembodiment, a carboxylic "arm" of an MRI contrast agent is used as achemically active functional group to attach the MRI agent to achemically functionalized optical dye, without a linker or spacer.Alternatively, there is a group between the MRI agent and optical dyethat serves to link the two together. In one embodiment, the linker is arelatively short coupling moiety, that is used to attach the two. Acoupling moiety may be synthesized directly onto an MRI agent forexample, and contains at least one functional group to facilitateattachment of the optical dye. Alternatively, the coupling moiety mayhave at least two functional groups, which are used to attach afunctionalized MRI agent to a functionalized optical dye. In anadditional embodiment, the linker is a polymer. In this embodiment,covalent attachment is accomplished either directly, or through the useof coupling moieties from the agent or dye to the polymer.

In a preferred embodiment, the covalent attachment is direct, that is,no linker is used. In this embodiment, the MRI contrast agent preferablycontains a carboxylic acid which is used for direct attachment to thefunctionalized optical dye, such as is depicted below in Structure 5.Thus, for example, for direct linkage to a carboxylic acid group of aMRI contrast agent such as DOTA or DTPA, amino modified or hydrazinemodified optical dyes will be used for coupling via carbodiimidechemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the optical dye, such as iscommercially available. Alternatively, the anhydride form of the MRIcontrast agent such as DTPA or DOTA may be coupled to the amino modifiedoptical dye, as is described herein.

Structure 5 depicts a direct covalent attachment between the MRIcontrast agent (in this embodiment, unsubstituted DOTA, although as willbe appreciated both substituted DOTA as well as other MRI agents such asunsubstituted or substituted DTPA may be used) and a functionalizedoptical dye. As will be appreciated by those in the art, other MRIagents may also be used. Additionally, Structure 5 depicts a carbonyllink between the optical dye and the MRI agent, which may include anester linkage or other linkages depending on the functionality of theoptical dye. ##STR7##

In a preferred embodiment, the bifunctional detection agents of theinvention utilize a linker. By "linker" herein is meant either acoupling moiety or a polymer. In general, the embodiments utilizing acoupling moiety result in bifunctional detection agents that are lessthan about 2-3 MW, with less than about 2 being preferred and less thanabout 1 being particularly preferred. Similarly, embodiments utilizingpolymers as the linkers result in bifunctional detection agents thatrange from about 2 MW to about 100 MW, with from about 2 to about 50 MWbeing preferred and from about 2 to about 10 being particularlypreferred.

The choice of the linker will effect the functionality of the opticaldye. In a preferred embodiment, the optical dye is attached to an aminegroup on a linker, which in turn is covalently attached to the MRIcontrast agent. In this embodiment, the optical dye is functionalizedwith an amine-reactive group, such as an isothiocyanate (set 4 of theMolecular Probes catalog, supra), a succinimidyl ester (set 5 of theMolecular Probes catalog, supra), a sulfonyl halide (set 6 of theMolecular Probes catalog, supra), or others. Particularly preferred arethe isothiocyanate derivatives, such as fluoroscein isothiocyanate(FITC), rhodamine X isothiocyanate (XRITC), and tetramethylrhodamineisothiocyanate (TRITC).

In an additional preferred embodiment, the optical dye is attached to athiol group on the linker, which is in turn covalently attached to theMRI contrast agent. In this embodiment, the optical dye isfunctionalized with a thiol-reactive group such as a haloacetylderivative (particularly iodoacetamides; set 1 of the Molecular Probescatalog), a maleimide (particular N-ethylmaleimide; set 2 of theMolecular Probes catalog), or others.

In a preferred embodiment, the linker is a coupling moiety. A "couplingmoiety" is capable of covalently linking two or more entities. One endof the coupling moiety is attached to the MRI contrast agent, and theother is attached to the optical dye. In a preferred embodiment, thecoupling moiety is attached first to either a MRI contrast agent or anoptical dye, and then a functional group of the coupling moiety is usedto attach the second moiety. In this embodiment, a functional group maynot be required to attach to the MRI agent, for example, and thus thecoupling moiety contains at least one functional group to facilitateattachment. Suitable functional groups include, but are not limited to,amines, thiols, and carboxylic acids. In another embodiment, thecoupling moiety is bifunctional, and utilizes a functional group forattachment to both the MRI agent and the optical dye.

The functional group(s) of the coupling moiety are generally attached toadditional atoms, such as alkyl or aryl groups, to form the couplingmoiety. Oxo linkers are also preferred. As will be appreciated by thosein the art, a wide range of coupling moieties are possible, and aregenerally only limited by the ability to synthesize the molecule and thereactivity of the functional group. For example, a preferred embodimentsynthesizes a MRI agent with a coupling moiety attached, to which theoptical dye is then subsequently attached via the functional group ofthe coupling moiety. For example, for DOTA derivatives, both nitrogensubstitution (Structure 2) or carbon substitution (Structure 1) of thecyclen ring backbone is possible with a wide variety of groups. See forexample U. S. Pat. Nos. 4,885,363 and 5,358,704 (nitrogen substitution)and Moi et al., J. Am. Chem. Soc. 110:6266-6267 (1988) (carbonsubstitution), and Chang et al., Applications of Methods andRadiochemistry, Gergamon Press: New York 1982, Lambrecht et al., Ed.

Generally, the coupling moiety comprises at least one carbon atom, dueto synthetic requirements; however, in some embodiments, the couplingmoiety may comprise just the functional group.

In a preferred embodiment, the coupling moiety comprises additionalatoms as a spacer. As will be appreciated those in the art, a widevariety of groups may be used. For example, a coupling moiety maycomprise an alkyl or aryl group substituted with one or more functionalgroups. Thus, in one embodiment, a coupling moiety containing amultiplicity of functional groups for attachment of multiple MRIcontrast agents and optical dyes may be used, similar to the polymerembodiment described below. For example, branched alkyl groupscontaining multiple functional groups may be desirable in someembodiments.

By "alkyl group" or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to 100 carbon atoms (C1 -C100), with a preferred embodiment utilizingfrom about 2 to about 50 carbon atoms (C2-C50), with about C2 throughabout C10 being preferred. Also included within the definition of analkyl group are cycloalkyl groups such as C5 and C6 rings, andheterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.Additional suitable heterocyclic substituted rings are depicted in U.S.Pat. No. 5,087,440, expressly incorporated by reference. In someembodiments, two adjacent R groups may be bonded together to form ringstructures together with the carbon atoms of the chelator, such as isdescribed in U.S. Pat. 5,358,704, expressly incorporated by reference.These ring structures may be similarly substituted.

By "aryl" group herein is meant aromatic rings such as phenyl andheterocyclic aromatic rings such as pyridine, furan, thiophene, pyrrole,indole and purine.

In addition to the functional group used to covalently attach thecontrast agents, the alkyl and aryl groups may be further substituted byeither additional functional groups or other atoms. For example, aphenyl group may be a substituted phenyl group. Suitable substitutiongroups include, but are not limited to, halogens such as chlorine,bromine and fluorine, amines, hydroxy groups, carboxylic acids, nitrogroups, carbonyl and other alkyl and aryl groups. Thus, arylalkyl andhydroxyalkyl groups are also suitable for use in the invention.

Preferred coupling moieties include, but are not limited to, alkyl andaryl amines, such as aminobenzyl (particularly p-aminobenzyl) andamino-benzyl derivatives, and alkyl and aryl thiols, and oxo groups.Particularly preferred bifunctional detection agents utilizing thesecoupling moieties are depicted below in Structures 6 (with otherwiseunsubstituted DOTA as the MRI agent, p-aminobenzyl as the couplingmoiety, and rhodamine as the optical dye), 7 (with otherwiseunsubstituted DOTA as the MRI agent, aminopropyl as the coupling moiety,and rhodamine as the optical dye) and 8 (with otherwise unsubstitutedDOTA as the MRI agent, methoxyamine as the coupling moiety, andrhodamine as the optical dye): ##STR8##

Thus, in a preferred embodiment, the bifunctional detection agents ofthe present invention utilize a coupling moiety, depicted herein as "X"with a covalently attached to an optical dye. The coupling moiety isattached either via a carboxylic "arm" of the MRI agent (exemplified byDOTA below, although any of the other MRI agents such as DTPA may alsobe used), as depicted in Structure 9, via a backbone carbon of thechelator of the MRI agent, as depicted in Structure 10, or at a backbonenitrogen, as depicted in Structure 11: ##STR9##

As noted above, the MRI agent may contain additional substitutiongroups.

Particularly preferred examples of this embodiment utilize DOTA as theMRI agent, p-aminobenzyl, methoxyamine, and aminobutyl as the X linker,and fluoroscein and rhodamine as the optical dye (isothiocyanatederivatives).

In a preferred embodiment, the linker comprises a polymer. As usedherein, a "polymer" comprises at least two or three subunits, which arecovalently attached. At least some portion of the monomeric subunitscontain functional groups for the covalent attachment of MRI contrastagents and optical dyes. In some embodiments coupling moieties are usedto covalently link the subunits with the MRI agent or optical dye.Preferred functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups, with amino groups beingparticularly preferred. As will be appreciated by those in the art, awide variety of polymers are possible. Suitable polymers includefunctionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the polymer contains a single type offunctional moiety for covalent attachment. In this embodiment, both theMRI contrast agent and the optical dye are attached using the samefunctionality. In this embodiment, as is outlined herein, some portionof the subunits contain MRI agents, some portion contains optical dyes,and generally some portion of the subunits do not contain either, as ismore fully described below. As will be described herein, in someinstances the unreacted functional groups are protected or "capped" toneutralize the functionality, i.e. from undesirable side reactions orexcess charge, as is described below.

In this embodiment, every monomeric subunit may contain the samefunctional moiety, or alternatively some of the subunits comprise afunctional moiety and others do not. Thus, for example, polylysine is anexample of a polymer in which every subunit comprises an aminofunctional group. Polyamino acids comprising lysine and alanine are anexample of polymers in which some of the subunits do not comprise achemically reactive functional moiety, as the alanine amino acids do notcontain a functional moiety that can be used to covalently attach eitherMRI agents or optical dyes, and thus do not need to be protected.

In a preferred embodiment, the polymer comprises different, i.e. atleast two, functional groups. Thus for example, polystyrene with aminoand thiol functional groups can be made or polyamino acids with twofunctional groups, such as polymers comprising lysine (ε-aminofunctional group) and glutamic acid (carboxy functional group). In thisembodiment, one functionality is used to add the MRI contrast agent andthe other is used to add the optical dye. Polymers can be generated thatcontain more than two functionalities as well.

In this embodiment, as described above, it is also possible toincorporate monomeric subunits that do not contain a functional moiety,for example, to avoid the use of protecting groups, i.e to decreasetoxicity as outlined herein.

The length of the polymer can vary widely. As will be appreciated bythose in the art, the size of the bifunctional detection agent willaffect the membrane permeability of the agent. Generally, the larger theagent, the less membrane permeable. However, bifunctional detectionagents which are quite large may tend to aggregate in cells over time,and thus may not be desirable in some embodiments. The size of thebifunctional detection agent will depend on the length of the polymerand the number of MRI agents and optical dyes per polymer. Generally,the bifunctional detection agents utilizing polymers range from about 4to about 50,000 MW, with from about 3,000 to about 30,000 beingpreferred, and from about 5,000 to about 25,000 being particularlypreferred.

The smallest polymer has two or three monomeric subunits, (n=2 or n=3)one of which has an MRI contrast agent covalently attached, and anotherof which has an optical dye covalently attached. Preferably, a thirdmonomeric subunit is between them, to minimize unnecessary stericinteractions, although this is not required. Generally, as outlinedabove, the length of the polymer is determined by the total molecularweight of the bifunctional detection agent, rather than the absolutenumber of monomeric subunits. However, preferred polymers include fromabout 10 to about 1000 monomeric subunits, with from about 20 to about500 being preferred and from about 30 to about 400 being particularlypreferred.

The number of MRI agents covalently attached per polymer can varywidely, and will depend in part on the synthetic conditions chosen.Without being bound by theory, it appears that to visualize anindividual cell using an MRI contrast agent, at least approximately1,500 Gd(III) ions are preferred. Thus, both the quantity of thebifunctional detection agent, and the amount of MRI contrast agentmolecules per polymer, can be varied to allow suitable amounts to beused. In general, the mole % of monomeric subunits containing covalentlyattached MRI agents can vary from less than 1 mole % to over 99 mole %,with from about 20 mole % to about 50 mole % being preferred, and fromabout about 10mole % to about 40mole % being particularly preferred.

Similarly, the number of optical dyes covalently attached per polymercan also vary widely. In general, the mole % of monomeric subunitscontaining covalently attached optical dyes can vary from less than 1mole % to over 99 mole %, with from about 20 mole % to about 50 mole %being preferred, and from about about 10mole % to about 40mole % beingparticularly preferred.

In a preferred embodiment, chemically reactive functional groups of thepolymer which do not contain either an MRI agent or an optical dye are"capped" or "protected" with a "protecting group" to neutralize thefunctionality. For example, polylysine may be toxic to cells, due to theexcessive charge; thus unreacted amino groups are preferablyneutralized. The choice of the protecting group will depend on thefunctionality to be neutralized. Protecting groups are well known in theart; see for example, Greene, Protecting Groups in Organic Synthesis,John Wiley & Sons, 1991, hereby incorporated by reference. When thefunctionality is an amino group, a preferred protecting group isβ-propiolactone.

In one embodiment, the bifunctional detection agents of the inventionthat utilize polymers may also utilize coupling moieties (depictedherein as X) to attach the polymers to either the MRI agents or theoptical dyes or both. That is, a coupling moiety may be used tofacilitate attachment of the MRI agents and/or optical dyes to thepolymer.

The bifunctional detection agents of the invention are synthesized asfollows.

Direct covalent attachment of an MRI agent and an optical dye may beaccomplished in several ways. A carboxylic acid group of a chelatingcomponent of an MRI contrast agent such as DOTA or DTPA is directlylinked to an amino modified or hydrazine modified optical dyes viacarbodiimide chemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the optical dye, such as iscommercially available. Alternatively, the anhydride form of the MRIcontrast agent such as DTPA or DOTA may be coupled to the amino modifiedoptical dye as is outlined in the Examples.

When a coupling moiety is used as a linker, the synthesis can proceed inseveral ways. In a preferred embodiment, the MRI contrast agent issynthesized containing the coupling moiety, and then the functionalgroup of the coupling moiety is used to attach the optical dye. Forexample, for DOTA derivatives, both nitrogen substitution (Structure 2)or carbon substitution (Structure 1) of the cyclen ring backbone ispossible with a wide variety of groups. See for example U.S. Pat. Nos.4,885,363 and 5,358,704 (nitrogen substitution) and Moi et al., J. Am.Chem. Soc. 110:6266-6267 (1988) (carbon substitution). Similarly, DTPAmay also be altered at either of these positions as well, see forexample Chang et al., supra.

When a polymer is used as the linker, the synthesis can proceed in anumber of ways, depending on the functional groups chosen. In apreferred embodiment, an excess of the anhydride form of the MRI agentsuch as DOTA or DTPA is reacted with a polymer containing an aminofunctionality such as polylysine. The amount of the excess will drivethe amount of MRI agent covalently attached to the polymer. Thus, as isoutlined in the examples, using a 50×excess of DTPA anhydride topolylysine of n=171 results in roughly 5 to 60 DTPA molecules perpolymer. A 100× excess results in from about 5 to about 20 DTPAs perpolymer, a 200× excess results in about 25 DTPAs, and a 400X excessresults in roughly 35 DTPAs per polymer. In this embodiment, theconcentration of the polylysine is kept low to avoid cross-linking thatmay occur at higher concentrations.

Alternatively, the MRI agents and/or optical dyes may be functionalizedor contain a coupling moiety, and then added to the polymer using thetechniques disclosed herein.

Once the MRI agent is covalently attached, the optical dye is attached,generally through the use of an isothiocyanate derivative if the polymercontains amino functionalities.

After the MRI agents and optical dyes are attached, unreacted functionalgroups may be protected, if necessary. For example, amino groups may beprotected using β-propyllactone, or other protecting groups. This may benecessary to detoxify the polymer. Thus, for example, the high charge ofpolylysine may render it toxic to cells; thus the excess charge can beneutralized.

Additionally, the charge of the bifunctional detection agent may bealtered for membrane permeability. Generally, charged molecules areimpermeable, with neutral compounds being significantly more permeable.In a preferred embodiment, the bifunctional detection agent is membraneimpermeable. Thus, in this embodiment, one of the carboxylic acid "arms"of either DOTA or DTPA is used for attachment, since in the presence ofthe Gd(III) this renders the MRI agent neutral.

Once made, the bifunctional detection agents of the invention find usein a number of applications. In a preferred embodiment, the bifunctionaldetection agents are used to image cells or tissues in the same way thatoptical dyes and MRI agents are used, as is well known in the art. Thus,they are administered either to a cell, tissue or organism and detectedusing known optical and MRI detection methods. Since MRI agents areoptically silent, these agents may be traced via the attachment to theoptical dye. The uses of these agents will be obvious to those skilledin the art.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 DOTA linked via a coupling moiety to rhodamine

I. General.

All solvents and reagents for organic synthesis were obtained fromAldrich or Fluka and used without further purification.Tetramethylrhodamine-5-isothiocyanate (TRITC) was obtained fromMolecular Probes. Distilled-deionized water was obtained from a NANOpurewater purification system and was used throughout to minimizetrace-metal contamination of the ligands and chelates. Reactions wereperformed in oven-dried flasks under positive argon pressure andmonitored by analytical thin-layer chromatography (TLC) using E.Mercksilica gel 60F plates (0.25 mm). E.Merck silica gel (230-400 mesh) wasused for flash chromatography. 1H and 13C NMR spectra were recorded at300 MHz on a GE300 NMR spectrometer. Analytical HPLC was performed witha Waters 600E Liquid Chromatograph using a reverse phase VYDAC 201HS544.6 mm×25 cm 5 micron C18 column. The samples were injected onto the C18reverse phase column and eluted with 100 mM triethylammonium acetatewith a gradient of 1-20% acetonitrile at a flow rate of 1 mL/min. Theproducts were detected by UV absorption at 260 and 330 nm. Thefluorescence of gadolinium-containing compounds was analyzed on aHitachi F-4500 instrument (excitation at 280 nm, emission ranged from313 to 335 nm). Spin-lattice relaxation time (T1) measurements wereobtained on a relaxometer operating at a fixed magnetic field. T1 wasdetermined for different Gd-complex concentrations (0.1-10 mM) in weaklybuffered medium (100 mM sodiumcarbonate) at varying pH (pH 7, 8 and 9).

II. Synthesis of various ligands

2-(p-Aminobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraaceticacid1 (PABD). The synthesis of PABA was performed as described in theliterature (McMurray et al., Bioconjugate Chem. 3:108-117 (1992);Garrity et al., Tet. Lett. 34:5531-5534 (1993); Takenouchi et al., J.Org. Chem. 58:6895-6899 (1993); Ansari et al., Bioorg. & Med. Chem.3:1067-1070 (1993); Renn et al., Bioconjugate Chem. 3:563-569 (1992).

Gd(PABD). Method A. To a stirred solution of p-aminobenzyl-DOTA ineither water or 100 mM sodiumcarbonate (pH 8) was addedgadoliniumchloride (0.99 eq). The reaction mixture was allowed to stirat 80° C. for 12 h. The solution was then concentrated to near dryness,redissolved in 100 mM triethylammoniumacetate/acetonitrile and purifiedby ETLC chromatography. Method B. To a stirred solution ofp-aminobenzyl-DOTA in either water or 100 mM sodiumcarbonate (pH 8) wasadded gadoliniumoxide (1.3 eq). The reaction mixture was allowed to stirat 80° C. for 18 h. The solution was filtered through an Acrodisc (LC 13PVDF, 0.2 mm) to remove unreacted gadoliniumoxide. The solution was thenconcentrated to near dryness, redissolved in 100 mMtriethylammoniumacetate/acetonitrile and purified by HPLCchromatography.

2-(4-Aminobutyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraaceticacid 2 (Lys-DOTA). The synthesis of Lys-DOTA was performed as describedin the literature (Crivici et al., Syn. Comm. 23:49-53 (1993); Cox etal., J. Chem. Soc. Perkin Trans. I 1990 p2567. ##STR10##

Ethyl-2,3-dibromopropionate 4 0.95 ml (6.53 mmol) was added to a mixtureof 4-nitrobenzylalcohol 5 (1 g, 6.53 mmol) and anhydrouspotassiumcarbonate 1.1 g (7.8 mmol) in anhydrous acetonitrile and thereaction mixture was stirred for 12 h at 70° C. The reaction mixture wasthen allowed to cool down to room temperature, concentrated to a smallervolume and diluted with dichloromethane. The combined organic layerswere washed with 0.5 N NaH₂ PO₄ solution and brine, and dried overmagnesiumsulfate. Concentration of the solution followed by purificationby flash chromatography (methylenechloride/hexane, 4:1) provided thedesired product 3-(4-nitrobenxyloxy)-2-bromopropionic acid 6.

To a stirred solution of cyclen 7 (100 mg, 0.58 mmol) was slowly added3-(4-nitrobenxyloxy)-2-bromopropionic acid 6 (128 mg, 0.39 mmol). Thesolution was stirred at 50° C. for 48 h. The resulting solution wasconcentrated to dryness and the residue was suspended in water,acidified with HCl to pH 2.5 and extracted with chloroform. The aqueousphase (neutralized by addition with 1N KOH) was loaded onto an AmberliteIR 120 cation-exchange-column (H+form). The column was first eluted withwater to neutrality followed by elution of the product 8 with 4N NH₄ OH.

To a stirred solution of the modified cyclen 8 (100 mg, 0.24 mmol) inwater was added a solution of bromoacetic acid 9 (197 mg, 1.42 mmol) andan equimolar amount of KOH (80 mg, 1.42 mmol) in water. The resultingsolution was brought to pH 10 with a 7M KOH solution and was stirred at80 C for 12 h. During this time the pH was maintained at 10 by addingadditional KOH solution. After 12 h the mixture was cooled to roomtemperature, and the product was isolated by acidification (pH 10) withconc. HBr. The crude product was filtered off and purified bycation-exchange chromatography and HPLC. The final product 3 wasobtained by quantitative reduction of the nitro-group to the amine usingLindlar catalyst in ethanol.

III. Coupling to an optical agent.

Gd(Rhoda-DOTA). To stirred solution of Gd(PABA) (1 mg, 1.46 mmol) in 250ml N,N-dimethylformamide was added TRITC (0.72 mg, 1.61 mmol, 1.1 eq).The solution was covered with aluminium foil and allowed to stir at roomtemperature for 10 h. The solution was then concentrated to dryness,taken up in 100 mM sodiumcarbonate/acetonitrile and purified by HPLCchromatography.

Example 2 DTPA linked via polylysine to rhodamine

Modification of Poly-d-lysine (pdl) with DTPA and rhodamine Pdl(4,25,53k) was purified by FPLC on a superdex 75 gel filtration column.Derivitized polymers of 7 to 36 DTPA molecules/polylysine were obtainedby successively increasing the excess of DTPA anhydride at ratios of100×, 200× and 400× DTPA anhydride to polylsine. The purified ratio of 1DTPA per 3-4 lysine monomer units was obtained regardless of themolecular weight of the polymer.

1) Attachment of DTPA to pdl (in 400 molar excess of DTPA to pdl): Atambient temperature, native pdl was dissolved with stirring in buffer(0.5 NaHCO₃, pH 9.7). DTPA anhydride was added slowly and allowed toreact for 45 minutes with stirring. The product was purified over gelfiltration (Sephadex 75) using a Pharmacia FPLC system and speedvaccedovernight.

2) Chelation of Gd³⁺ to TRITC-G-DTPA-pdl:

In a 75° C. oil bath with stirring, GdCl₃ (0.1 ml of a 5.3 mg/mlsolution of GdCl₃ in H₂ O) was added slowly to TRITCG-DTPA-pdl compounddissolved in H₂ O. The reaction proceeded for 1 hour, then was purifiedvia gel filtration using a Sephadex G-25 PD 10 column pre-equilibratedwith water and overnight.

3) Attachment of TRITC-G to DTPA-pdl (in 30 molar excess of TRITC-G toDTPA-pdl):

At ambient temperature, Gd-DTPA-pdl was dissolved in a minimum of H₂ Oor DMF. TRITC-G was dissolved in DMF (or H₂ O) (0.5 ml DMF for 5 mgsTRITC-G) to form an opaque slurry. This slurry was added over 10 minuteswith stirring to the DTPA-pdl, along with a 0.2 ml of rinse of buffer.The reaction proceeded with stirring for 2 hours. The product waspurified via gel filtration using a Sephadex G-25 PD 10 columnpre-equilibrated with water, then speedvacced overnight.

4) β-propiolactone capping

In order to render the double score agent nontoxic when injecteddirectly into cells the GdDTPA-Rhodamine-Poly-d-Lys was reacted withβ-propiolactone with only minor modification to a published procedure(see U.S. Pat. No. 3,907,755. This reaction efficiently transforms theεnitrogens of the lysine derivatives to OH groups, and subsequenttoxcity studies confirmed the success of the procedure.

Xenopus lavevis embryo at stage 8 was injected with GRIP into themarginal blastomere at the 16 cell stage. The labelled cells populatethe prosepective mesodermal regions as seen in confocal fluorescenceimage (data not shown). The distribution of labelled cells is confirmedusing MRI and delineate cells that are unobservable by fluorscencetechniques (data not shown). The progeny of the labeled blastomere at 5days later at stage ·38-39 were imaged (data not shown), and labeledcells in somitic mesoderm were clearly visible and span the length ofeach somitic block as expected. Several cells in endoderm are alsolabeled, which is in agreement with published fate maps of 16 cell stageembryos.

I claim:
 1. A bifunctional detection agent comprising a polymercovalently linked to at least one optical dye and at least one MRIcontrast agent.
 2. A polymer comprising a first subunit covalentlyattached to an MRI agent, and a second subunit covalently attached to anoptical contrast agent.
 3. A polymer according to claim 2 furthercomprising a third subunit.
 4. A polymer according to claim 2 whereinsaid first and second subunits are the same.
 5. A polymer according toclaim 2 wherein said first and second subunits are different.
 6. Abifunctional detection agent according to claim 1 or 2 wherein saidoptical dye is selected from the group consisting of fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, and Texas Red.
 7. A bifunctional detection agentaccording to claim 6 wherein said optical dye is fluorescein.
 8. Abifunctional detection agent according to claim 6 wherein said opticaldye is rhodanine.
 9. A bifunctional detection agent according to claim 1or 2 wherein said MRI contrast agent is selected from the groupconsisting of 1,4,7,10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid(DOTA), substituted DOTA, diethylenetriaminepentaacetic (DTPA),substituted DTPA, 1,4,7,10-tetraazacyclododecane-N,N', N",N'"tetraethylphosphorus (DOTEP), substituted DOTEP,1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (Do3A), andsubstituted Do3A.
 10. A bifunctional detection agent according to claim9 wherein said MRI contrast agent is DOTA.
 11. A bifunctional detectionagent according to claim 9 wherein said MRI contrast agent is DTPA. 12.A bifunctional detection agent according to claim 1 or 2 wherein saidpolymer is a functionalized dextran.
 13. A bifunctional detection agentaccording to claim 1 or 2 wherein said polymer is a polyamino acid. 14.A bifunctional detection agent according to claim 13 wherein saidpolyamino acid is polylysine.
 15. A bifunctional detection agentaccording to claim 1 or 2 wherein said polymer has a molecular weight ofless than 40 kD.
 16. A bifunctional detection agent according to claim 1or 2 wherein said polymer has a molecular weight of less than 25 kD. 17.A bifunctional detection agent according to claim 1 or 2 wherein saidpolymer has a molecular weight of less than 15 kD.
 18. A bifunctionaldetection agent according to claim 1 or 2 wherein said polymer has amolecular weight of less than 10 kD.
 19. A method of visualizing a cell,tissue or organism comprising administering a bifunctional detectionagent according to claim 1 or 2 and optically visualizing said cell,tissue or organism.
 20. A method of visualizing a cell, tissue ororganism comprising administering a bifunctional detection agentaccording to claim 1 or 2 and visualizing said cell, tissue or organismwith magnetic resonance imaging.